Compositions and methods for preventing erythropoietin-associated hypertension

ABSTRACT

The inventors have discovered that both soluble erythropoietin-binding protein and antibodies against the erythropoietin-binding protein, when they are administered to a mammal along with erythropoietin (Epo), prevent or reduce the blood pressure increase normally caused by erythropoietin, while not affecting the hematocrit increase that is the purpose of Epo treatment. The invention provides a method of treating anemia in a mammal involving: administering erythropoietin (Epo) to the mammal; and administering to the mammal an agent selected from a soluble Epo-binding protein (Epo-bp), a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor, and a combination thereof. The invention also provides a method of reducing hypertension in a mammal receiving Epo, and pharmaceutical compositions containing a soluble Epo-bp and/or a recognition protein that binds Epo receptor on an extracellular soluble portion of the Epo receptor

BACKGROUND

Erythropoietin is sold under the labels PROCRIT (epoetin), EPOGEN(epoetin), and ARANESP (darbepoetin). Erythropoietin is indicated fortreatment of anemia, particularly anemia associated with chronic renalfailure and cancer chemotherapy.

Erythropoietin (Epo), an angiogenic factor, increases hematocrit andhemoglobin concentrations via the stimulation of erythropoiesis,resulting in increased blood viscosity (1-5) and blood pressure (1-14).In clinical studies, approximately one-third of hemodialysis patientstreated with recombinant human Epo have shown an increase in bloodpressure. Epo has been postulated to increase peripheral vascularresistance and decrease cardiac output due to increased viscosity (6).Others have suggested additional mechanisms for Epo-caused hypertension,such as hypervolemia, increased plasma renin and angiotensin, along withadrenergic overactivity, increased plasma arginine vasopressin,decreased kinins and prostaglandins (15). An excessive, lastingelevation of circadian amplitude, blood pressure overswinging and anelevation of blood pressure may be based on vasomotor chronome (timestructure) alteration. Hormones and other agents, in part on a geneticbasis, may be contributing factors to the circadian blood pressurevariation.

Hypertension is one of the most important risk factors in thedevelopment of cardiovascular complications. Hypertension is affectedsignificantly by circadian rhythms. Hormonal concentration in the bodyfluctuates during the day and night with prominent spontaneous circadian(about-24-hour) changes that affect blood pressure and heart rate. Thereare also sufficiently important rhythms that can make the differencebetween the stimulation versus the inhibition of a malignancy.

Epo is a 34 kDa glycoprotein hormone that is produced by theinterstitial cells in the peritubular capillary bed of the mammaliankidney and the perivenenous hepatocytes of the liver (3). Epo issecreted in response to hypoxia to coordinate erythropoiesis as aprimary inducer and regulator of red cell differentiation by suppressingapoptosis and triggering cell division and terminal maturation of bloodcell progenitors (16). These effects are mediated through the binding ofEpo to specific cell surface receptors (17). The primary structure ofhuman Epo has been known since the mid-1980s (18,19), but the structuralfeatures of the Epo molecule that confer its biological activity arelargely unknown. Human Epo contains two disulfide bonds that linkcysteine 29 with cysteine 33, and cysteine 7 with cysteine 161. Bothbonds are essential for biological activity (18). Epoetin (recombinanthuman erythropoietin) was produced following isolation of the human geneand its expression in a Chinese hamster's ovarian cell line (4). Therecombinant Epo is a 165-amino acid mature protein that differs from themature native protein only in lacking the carboxyl terminus arginine ofthe native protein. Native human Epo is translated as a 193-amino acidpeptide, from which a 27-amino-acid leader sequence is cleaved off (19,20). Recombinant Epo has an apparent molecular weight of about 30.4 kDa,appears to be immunologically equivalent to the endogenous hormone, andexhibits full biological activity (19).

Epo-treated humans and animals exhibit increased hematocrit % andincreased hemoglobin via the stimulation of erythropoiesis (2-5). Somestudy results suggest that increased hematocrit levels are correlatedwith an increased blood pressure in humans (20). Other studies involvingthe treatment of anemia with Epo showed increased hematocritconcentrations and resulting elevated blood severe enough to requiretreatment with antihypertensive medication pressure in 20-30% ofpatients (5).

Hypertension is the most frequent and most important complication fromtreatment with erythropoietin. Furthermore, although the goal of Epotreatment is to increase hematocrit and hemoglobin, it has been foundthat the greater the increase of hematocrit, the greater the risk ofmortality and cardiovascular events (PROCRIT warnings,www.rxlist.com/cgi/generic/epoetin-wcp.htm). This may be due to bloodpressure rise, since the extent of blood pressure rise has been shown tocorrelate with the extent of hematocrit increase (20). The epoetin labelwarns that patients with uncontrolled hypertension should not be treatedwith epoetin.

New methods and compositions to prevent or treat blood pressure rise inpatients treated with Epo are needed. New methods and compositions totreat anemia without causing hypertension are needed.

SUMMARY

The inventors have discovered that a soluble Epo-binding protein, whichis a soluble fragment of the membrane protein Epo receptor, whenadministered to mammals along with Epo, prevents the blood pressureincrease ordinarily caused by. Epo, while not affecting the rise inhemoglobin and hematocrit that is the goal of Epo treatment. An antibodyagainst Epo-binding protein was also found to prevent the Epo-causedblood pressure increase while not affecting the Epo-induced hematocritrise.

Accordingly, the invention provides a method of treating anemia in amammal involving: administering erythropoietin (Epo) to the mammal; andadministering to the mammal an agent selected from a soluble Epo-bindingprotein (Epo-bp), a recognition protein that binds Epo receptor on anextracellular soluble portion of the Epo receptor, and a combinationthereof.

Another embodiment of the invention provides a method of reducinghypertension in a mammal receiving Epo involving administering to themammal an agent selected from a soluble Epo-binding protein (Epo-bp), arecognition protein that binds Epo receptor on an extracellular solubleportion of the Epo receptor, and a combination thereof.

Another embodiment of the invention provides use of a recognitionprotein that binds Epo receptor on an extracellular soluble portion ofthe Epo receptor in medical therapy.

Another embodiment of the invention provides use of a recognitionprotein that binds Epo receptor on an extracellular soluble portion ofthe Epo receptor to prepare a medicament effective to reduceerythropoietin-induced hypertension.

Another embodiment of the invention provides use of a solubleerthyropoietin-binding protein in medical therapy.

Another embodiment of the invention provides use of a solubleerythropoietin-binding protein to prepare a medicament effective toreduce erythropoietin-induced hypertension.

Another embodiment of the invention provides a pharmaceuticalcomposition including: erythropoietin; and an agent selected from asoluble Epo-binding protein (Epo-bp), a recognition protein that bindsEpo receptor on an extracellular soluble portion of the Epo receptor,and a combination thereof.

Another embodiment of the invention provides a pharmaceuticalcomposition including: a recognition protein that binds Epo receptor onan extracellular soluble portion of the Epo receptor.

Another embodiment of the invention provides a pharmaceuticalcomposition including: a soluble Epo-binding protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the average circadian blood pressure ofeach of the treatment groups of rats treated with Epo and other agents.Error bars represent standard error, SEM.

FIG. 2 shows the circadian blood pressure measurements for each of thetreatment groups.

FIG. 3 is a representation of the circadian hematocrit variation in thetreatment groups of rats.

FIG. 4 is the outline of photographs of spleens isolated from ratstreated with Epo (panels A, B, and C), or saline (panel D).

FIG. 5 is a bar graph of optical density from immunodetection of Epo,αEpo, Epo-bp, and αEpo-bp in serum and plasma of human volunteers. Errorbars represent standard error, SEM.

DETAILED DESCRIPTION

Definitions.

“Erythropoietin” as used herein includes erythropoietin isolated fromnatural sources and recombinant or engineered erythropoietin that hasthe biological activity of erythropoietin of stimulating red blood cellproduction. It includes epoetin and darbepoetin. Preferably, theerythropoietin has at least 70%, more preferably at least 90%, aminoacid sequence identity with human erythropoietin, SEQ ID NO:3. Sequenceidentity is calculated using the default BLAST parameters for nucleotidesequence comparison at the PubMed website, www.ncbi.nlm.nih.gov/PubMed/.

As used herein, “a soluble Epo-binding protein” refers to a protein thatis not an antibody, is water-soluble, binds erythropoietin with highaffinity, and when administered with Epo to mammals is effective atreducing an Epo-induced blood pressure rise in the mammals. Preferably,the K_(D) of the protein for binding with erythropoietin is less than 10μM, more preferably less than 1 μM, more preferably still less than 100nM, and most preferably less than 20 nM. K_(D) can be determined bycompetition binding assays such as described in Example 6 of U.S. Pat.No. 5,843,726. Preferably, the soluble Epo-binding protein is orincludes sequences from or sequences homologous to the soluble portionof an Epo receptor. The human Epo receptor sequence is SEQ ID NO:1(Winkelmann, J. C., et al., 1990, Blood 76: 24-30). The soluble portionof the human Epo receptor is residues 25-250 of SEQ ID NO:1. In aparticular embodiment, the residues of the Epo-binding proteinresponsible for binding Epo are at least 70% identical, more preferablyat least 80% identical, more preferably at least 90% identical, mostpreferably identical, to the corresponding residues of SEQ ID NO: 1.

As used herein, “an extracellular soluble portion of an Epo receptor”refers to the portion of the Epo receptor that is exposed on theextracellular surface of the cell in the aqueous environment.Specifically, it refers to SEQ ID NO:2, which is residues 25-250 of SEQID NO:1 (the human Epo receptor), or to the homologous soluble residuesof another Epo receptor protein.

As used herein, “a recognition protein that binds Epo receptor on anextracellular soluble portion of the Epo receptor” refers to a proteinthat binds the extracellular soluble portion of Epo receptor and that,when administered to mammals along with Epo, reduces an Epo-inducedblood pressure rise in the mammals. The recognition protein can be acomplete antibody raised against an Epo receptor or against anEpo-binding protein, where the antibody binds the soluble portion of Eporeceptor, or a binding fragment of such a complete antibody. Therecognition protein can also be a non-antibody protein or peptide (e.g.,a protein or peptide selected by phage display binding) that binds tothe extracellular soluble portion of the human Epo receptor or ofanother mammalian Epo receptor with a binding affinity of at least 10⁵liters per mole, more preferably 10⁶, more preferably at least 10⁷, mostpreferably at least 10⁸ liters per mole.

As used herein, the term “antibody” includes complete antibodies andantigen-binding fragments of complete antibodies, e.g., Fab or F(ab′)₂antibodies. The term “1antibody” also includes both monoclonal andpolyclonal antibodies (e.g., antiserum).

The term “reducing hypertension” by administering an agent includespreventing or reducing an increase in blood pressure that otherwiseoccurs in a significant fraction of a population when the agent is notadministered.

DESCRIPTION

The methods of the invention involve administering to the mammal anagent selected from a soluble Epo-binding protein (Epo-bp), arecognition protein that binds Epo receptor on an extracellular solubleportion of the Epo receptor, and a combination thereof.

In some embodiments of the invention, the agent is a soluble Epo-bp.

In some embodiments, the soluble Epo-bp contains a fragment of a solubleportion of a mammalian Epo receptor.

In particular embodiments the soluble Epo-bp comprises a fragment of atleast 30 residues of SEQ ID NO:2 (residues 25-250 of human Epo receptor,SEQ ID NO:1). SEQ ID NO:2 is the extracellular soluble portion of thehuman Epo receptor. In other particular embodiments, the soluble Epo-bpcomprises a fragment of at least 15, at least 50, at least 100, at least150, or at least 200 residues of SEQ ID NO:2.

In particular embodiments, the soluble Epo-bp includes or is SEQ IDNO:2. The soluble Epo-bp that is SEQ ID NO:2 can be expressed asdescribed in U.S. Pat. No. 5,843,726. In general terms, SEQ ID NO:2 isexpressed as a fusion protein with a glutathione S-transferase (GST)N-terminal leader sequence. SEQ ID NO:2 is separated from the GST leadersequence by a thrombin cleavage site. The expressed fusion protein iscleaved with thrombin to release SEQ ID NO:2. The Epo-bp of SEQ ID NO:2is found naturally in human serum and plasma, possibly produced as acleavage product of Epo receptor (see Example 2 below).

In particular embodiments, the soluble Epo-bp has at least 70%, at least80%, or at least 90% amino acid sequence identity to SEQ ID NO:2, ascalculated using the default BLAST parameters for nucleotide sequencecomparison at the PubMed website, www.ncbi.nlm.nih.gov/PubMed/.

In some embodiments of the invention, the soluble Epo-bp is SEQ ID NO:8,which is SEQ ID NO:2 with the additional two residues Gly-Ser at theamino terminus.

In one embodiment of the invention, the soluble Epo-bp is a product of aprocess comprising: expressing a fusion protein and cleaving it withthrombin. The fusion protein consists essentially of a first polypeptidesegment having a thrombin proteolytic cleavage site at its carboxylterminus, and a second polypeptide segment consisting essentially of SEQID NO:2. The amino terminus of the second segment is covalently coupledto the carboxyl terminus of the first segment. The soluble Epo-bp isproduced by cleaving the fusion protein with thrombin.

In one embodiment of the invention, the soluble Epo-bp is a product of aprocess comprising: expressing a fusion protein comprising SEQ ID NO:2linked at its amino terminus to a peptide sequence ofLeu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:7), and cleaving the fusion proteinwith thrombin.

In one embodiment of the invention, the soluble Epo-bp is a product of aprocess comprising: expressing a fusion protein consisting of: (a) afirst polypeptide segment having an amino terminus and a carboxylterminus, said segment having SEQ ID NO:7 at its carboxyl terminus; and(b) a second polypeptide segment consisting of SEQ ID NO:2, the secondpolypeptide segment covalently coupled to the carboxyl terminus of thefirst polypeptide segment; and cleaving the fusion protein withthrombin.

In other particular embodiments of the methods of the invention, theagent is a recognition protein that binds Epo receptor on anextracellular soluble portion of the Epo receptor.

The recognition protein may exert its effect of reducing the Epo-inducedblood pressure increase by binding to the extracellular soluble portionof intact Epo receptor molecules in membranes, or it may exert itseffect by binding to the soluble Epo-binding protein that existsnaturally circulating in blood (which has the same amino acid sequenceas the extracellular soluble portion of the Epo receptor, and may be aproteolytic product of the receptor), or by both of these mechanisms orother unknown mechanisms. Describing this embodiment of the agent as “arecognition protein that binds Epo receptor on an extracellular solubleportion of the Epo receptor” is intended to describe a characteristic ofthe recognition protein, and not to necessarily describe the mechanismof action of the recognition protein.

In a particular embodiment, the recognition protein binds SEQ ID NO:2.That is, the recognition protein recognizes and binds to some sequencewithin SEQ ID NO:2. The recognition protein could, for instance, be anantibody raised against SEQ ID NO:2, an antibody raised against the Eporeceptor where the antibody binds to SEQ ID NO:2, or an antibody raisedagainst a peptide fragment of SEQ ID NO:2.

In particular embodiments, the recognition protein is an antibody. Inparticular embodiments, the antibody is a complete antibody. Inparticular embodiments, the antibody is an antibody fragment. Forinstance, the antibody fragment may be an Fab, Fab′, or F(ab′)₂, or Fv.

In particular embodiments, the antibody is an antibody against SEQ IDNO:2.

In other particular embodiments, the recognition protein is anon-antibody protein or peptide. For instance, it can be a recognitionpeptide or protein selected by phage display. Methods for selection ofbinding peptides using phage display are disclosed in Sidhu S S, LowmanH B, Cunningham B C, and Wells J A: Phage display for selection of novelbinding peptides. Methods in Enzymology 2000; 328:333-363.

In a particular embodiment, the agent is a combination of a solubleEpo-binding protein and a recognition protein that binds Epo receptor onan extracellular soluble portion of the receptor.

Epo and the agent may be administered separately or together.

In particular embodiments, the amount of the agent administered is atleast equimolar with the amount of Epo administered.

In particular embodiments, the amount of the agent administered is aboutequimolar with the amount of Epo administered. For instance, the motesof the agent administered may be between 75% and 125% of the mole of Epoadministered.

In particular embodiments of the method of treating anemia, the agentreduces an erythropoietin-induced blood pressure rise in the mammal.That is, the blood pressure of the mammal rises less when the mammalreceives Epo and the agent, than when the mammal receives Epo alone.

Preferably, when Epo is administered to a mammal with an equimolaramount of the agent, blood pressure increases no more than 75% as muchas it rises when Epo is administered alone to the mammal, morepreferably no more than 50% as much as it increases when Epo isadministered alone to the mammal.

One embodiment of the invention is a pharmaceutical compositioncontaining a recognition protein that binds Epo receptor on anextracellular soluble portion of the Epo receptor.

Another pharmaceutical composition of the invention includeserythropoietin; and an agent selected from a soluble Epo-bindingprotein, a recognition protein that binds Epo receptor on anextracellular soluble portion of the Epo receptor, and a combinationthereof.

Another pharmaceutical composition of the invention includes a solubleEpo-binding protein.

Typically, the pharmaceutical compositions include a pharmaceuticallyacceptable diluent or carrier.

In one embodiment of the pharmaceutical compositions containing therecognition protein, the recognition protein is an antibody against SEQID NO:2.

In one embodiment of the pharmaceutical compositions containing thesoluble Epo-binding protein, the Epo-binding protein is SEQ ID NO:2.

Other particular embodiments of the agent, the soluble Epo-bindingprotein, and the recognition protein that binds Epo receptor on anextracellular soluble portion of the Epo receptor are as described forthe methods of the invention.

Raising Antibodies

To generate antibodies, Epo receptor or Epo-bp can be administereddirectly to a mammal, or the proteins or peptide fragments thereof canbe coupled to a carrier protein. Suitable carrier proteins includekeyhole limpet hemocyanin, bovine serum albumin, and ovalbumin. Methodsof coupling to the carrier protein include single step glutaraldehydecoupling and other methods disclosed in Harlow, Ed et al., Antibodies: alaboratory manual, Cold Spring Harbor Laboratory (1988).

The immunogen is used to immunize a vertebrate animal in order to inducethe vertebrate to generate antibodies. Preferably the immunogen isinjected along with an adjuvant such as Freund's adjuvant, to enhancethe immune response. Suitable vertebrates include rabbits, mice, rats,hamsters, goats, sheep, and chickens.

Hybridomas to synthesize monoclonal antibodies can be prepared bymethods known in the art. See, for instance, Wang, H., et al., AntibodyExpression and Engineering, Am. Chem. Soc., Washington, D.C. (1995).Polyclonal and monoclonal antibodies can be isolated by methods known inthe art. See, for instance, id. and Harlow et al.

Native antibodies are tetramers of two identical light (L) chains andtwo identical heavy (H) chains. The L and H chains each have variabledomains that are responsible for antigen recognition and binding. Thevariability in the variable domains is concentrated in thecomplementarity determining regions (CDRs).

An antibody that is contemplated for use in the present invention can bein any of a variety of forms, including a whole immunoglobulin, anantibody fragment such as Fv, Fab, and similar fragments, a single chainantibody that includes the CDR, and like forms, all of which fall underthe broad term “antibody” as used herein.

The term “antibody fragment” refers to an antigen-binding portion of afull-length antibody. Antibody fragments can be as small as about 4amino acids, about 1 0 amino acids, or about 30 amino acids or more.Some types of antibody fragments are the following:

(1) Fab is the fragment that contains a monovalent antigen-bindingfragment of an antibody molecule. A Fab fragment can be produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain. Two Fab fragments areobtained per whole antibody molecule.

(2) Fab′ is the fragment of an antibody that can be obtained by treatingwhole antibody with pepsin, followed by reduction to yield an intactlight chain and a portion of the heavy chain. Two Fab′ fragments areobtained per whole antibody molecule. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxyl terminus ofthe heavy chain CH 1 domain including one or more cysteines.

(3) F(ab′)₂ is the fragment that can be obtained by digestion of wholeantibody with pepsin, without reduction. F(ab′)₂ is a dimer of two Fab′fragments held together by two disulfide bonds.

(4) Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. Fv consists of a dimer of one H and one Lchain variable domain in a tight, non-covalent association (V_(H)-V_(L)dimer). It is in this configuration that the three CDRs of each variabledomain interact to define an antigen-binding site. Collectively, the sixCDRs confer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to bind antigen, although at alower affinity than the complete binding site.

(5) A single chain antibody (SCA) is defined as a genetically engineeredmolecule containing the variable region of the light chain and thevariable region of the heavy chain linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

The preparation of polyclonal antibodies is well known to those skilledin the art. See, for example, Coligan et al., in Current Protocols inImmunology, section 2.4.1 (1992). The preparation of monoclonalantibodies is likewise conventional. See, for example, Harlow et al.,page 726.

Methods of in vitro and in vivo manipulation of monoclonal antibodiesare well known to those skilled in the art. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,Nature 256:495 (1975), or may be made by recombinant methods, e.g., asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies for usewith the present invention may also be isolated from phage antibodylibraries using the techniques described in Clarkson et al., Nature352:624 (1991), as well as in Marks et al., J. Mol. Biol. 222:581(1991). Another method involves humanizing a monoclonal antibody byrecombinant means to generate antibodies containing human specific andrecognizable sequences. See, for review, Holmes et al., J. Immunol.158:2192 (1997) and Vaswani et al., Annals Allergy, Asthma & Immunol.81:105 (1998).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) are identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Nat'l. Acad. Sci. 81:6851 (1984)).

Methods of making antibody fragments are also known in the art (see, forexample, Harlow and Lane, Antibodies: a Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988)). Antibody fragments of the presentinvention can be prepared by proteolytic hydrolysis of the antibody orby expression in E. coli of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)₂. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5 S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab fragments and an Fcfragment directly. These methods are described, for example, in U.S.Pat. Nos. 4,036,945, and 4,331,647, and references contained therein.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody. For example, Fv fragments comprise anassociation of V_(H) and V_(L) chains. This association may benoncovalent or the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde.Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains connected by an oligonucleotide.The structural gene is inserted into an expression vector, which issubsequently introduced into a host cell such as E. coli. Therecombinant host cells synthesize a single polypeptide chain with alinker bridging the two V domains. Methods for producing sFvs aredescribed, for example, by Whitlow et al., Methods: a Companion toMethods in Enzymology, 2:97 (1991); Bird et al., Science 242:423 (1988);Ladner et al., U.S. Pat. No. 4,946,778; and Pack et al., Bio/Technology11:1271 (1993).

Another form of an antibody fragment is a peptide containing a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) are often involved in antigen recognition andbinding. CDR peptides can be obtained by cloning or constructing genesencoding the CDR of an antibody of interest. Such genes are prepared,for example, by using the polymerase chain reaction to synthesize thevariable region from RNA of antibody-producing cells. See, for example,Larrick et al., Methods: a Companion to Methods in Enzymology, 2:106(1991).

The invention contemplates human and humanized forms of non-human (e.g.,murine) antibodies. Such humanized antibodies are chimericimmunoglobulins, immunoglobulin chains, or fragments thereof (such asFv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, goat, sheep, or rabbit having thedesired specificity, affinity, and capacity.

In some instances, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are found neither in the recipientantibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, humanized antibodies will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., Nature 321:522(1986); Reichmann et al., Nature 332:323 (1988); Presta, Curr. OpinionStruct. Biol. 2:593 (1992); Holmes et al., J. Immunol. 158:2192 (1997);and Vaswani et al., Annals Allergy, Asthma & Immunol. 81:105 (1998).

Antibodies of the invention can also be mutated to optimize theiraffinity, selectivity, binding strength or other desirable property. Onemethod of mutating antibodies involves affinity maturation using phagedisplay. Affinity maturation using phage display refers to a processdescribed in Lowman et al., Biochemistry 30:10832(1991); see alsoHawkins et al., J. Mol. Biol. 254:889(1992).

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES Example 1

The Example describes the preparation of an Epo-bp thought to have SEQID NO:2, as is also described in U.S. Pat. No. 5,843,726.

Construction of EpoR recombinant vector. A recombinant plasmidexpression vector, pJYL26, was constructed from a PCR product having thehuman Epo receptor extracellular soluble domain coding sequence and fromthe plasmid vector pGEX-2T, which was purchased from Pharmacia.

PCR amplification was carried out using a full-length human EpoR cDNA,SEQ ID NO:4, as a template. The 5′-sense primer was5′-TTGGATCCGCGCCCCCGCCTAAC-3′ (SEQ ID NO:5). This primer has a BamH1linker sequence at the 5′ end followed by the coding sequence for aminoacids 25-29 of the full-length human EpoR protein. The 3′-antisenseprimer was 5′-TGAATTCGGGGTCCAGGTCGCT-3′ (SEQ ID NO:6). This primer hasan EcoR1 linker followed by sequence complementary to the codingsequence for amino acids 250 through 246 of full-length EpoR. PCR wascarried out as described in U.S. Pat. No. 5,843,726.

The PCR product and pGEX-2T were digested with EcoR1 and BamH1, thedigested DNAs were purified with gel electrophoresis. The ligation wasdone with a mixture containing 1 μg/μl each of the PCR product andpGEX-2T. The ligated product was verified to be ˜5.5 kb. The ligatedplasmid mixture was used to transform E. coli JM109. Colonies weregrown. DNA was extracted from each transformed colony, and analyzed.Plasmid from one colony was selected after examining both EcoR1 andEcoR1 plus BamH1 digested DNA sizes in 1% agarose gels to confirm thepredicted sizes. The procedures were carried out as described in U.S.Pat. No. 5,843,726.

Purification of EpoRex-th fusion protein. Transformed E. coli containingthe recombinant vector pJYL26 was grown and induced with IPTG. Cellextract was passed through a GSH-agarose column. The bound EpoRex-th waseluted with reduced glutathione. This was performed as described in U.S.Pat. No. 5,843,726.

Purification of Epo-bp. EpoRex-th contains a thrombin-specificproteolytic cleavage recognition sequence separating Epo-bp fromglutathione-S-transferase. The amino acid sequence of the cleavagerecognition sequence is Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO:7). Thefusion protein was cleaved with thrombin, and Epo-bp was purified byaffinity binding to an Epo-agarose column. This was performed asdescribed in U.S. Pat. No. 5,843,726. The amino terminus of the Epo-bpproduced by this procedure, and thus the thrombin cleavage site, was notexperimentally determined. The thrombin cleavage is believed to produceEpo-bp of SEQ ID NO:2. But, thrombin may cleave at other sites, such asbetween the Arg and the Gly of the recognition sequence to produce aprotein having the Gly-Ser peptide attached to the amino terminus of SEQID NO:2 (SEQ ID NO:8).

Example 2

This Example describes experiments testing the effect of administeringEpo-bp, which is a soluble Epo-binding protein having the amino acidsequence of SEQ ID NO:2, and an Fab antibody against Epo-bp, either withor without Epo, to rats.

EXPERIMENTAL PROCEDURES

Materials:

Glutathione (GSH)-agarose, pGEX-2T expression vector and SEPHADEX G-50were purchased from Pharmacia (Mechanicsburg, Pa.). PCR reagents werefrom Perkin-Elmer Cetus (Norwalk, Conn.) and AFFIGEL® 15 was from BioRad(Richmond, Calif.). Bacteriophage T4 DNA ligase, restriction enzymes andisopropylthio-β-D-galactoside (IPTG) were purchased from BRL Gibco(Gaithersburg, Md.). GENECLEAN II was from Bio 101, La Jolla, Calif.Nitrocellulose was from Schleicher & Schuell Co. (Keene, N.H.).Chemiluminescence (ECL) reagents and ¹²⁵I-Epo were from Amersham(Arlington Heights, Ill.) and unlabeled Epo was a gift of Chugai-Upjohn(Rosemont, Ill.). Thrombin, trypsin, phenylmethylsulfonylfluoride(PMSF), diisopropylfluorophosphate (DFP), TRITON X-100,2,7-Dichlorofluoresein, biotin-amidocaproyl hydroazide, alkalinephosphatase conjugate, disodium p-nitrophenyl phosphate, ando-phenylenediaminedihydrochloride (oPD) were from Sigma (St. Louis,Mo.). Biotinylated rabbit anti-sheep antibodies, Avidin-horse radishperoxidase, and IgG purification Kit were from Pierce Co. (Rockford,Ill.). Streptavidin peroxidase was purchased from Boehringer ManheimCorp (Indianapolis, Ind.), and microplates were from Corning Costa(Cambridge, Mass.). All other chemicals were of reagent grade.

Epo-bp, Fab antibody against Epo-bp (αEpo-bp), and Fab antibody againstEpo (αEpo) were prepared in our laboratory. Epo-bp was prepared asdescribed in U.S. Pat. No. 5,843,726. Epo-bp had the amino acid sequenceof SEQ ID NO:2. A full-length human EpoR cDNA (SEQ ID NO:4) was from Dr.Bernard G. Forget, Yale University. Oligonucleotides were synthesized bythe microchemical facility of the Institute of Human Genetics,University of Minnesota, Minneapolis. All other chemicals were ofreagent grade.

Animal Study:

Male Sprague-Dawley (SD) rats were housed at the University Animal Carefacility with Purina Chow and drinking water freely accessible. Weexamined any circadian stage-dependence of Epo effects on the bloodpressure, hematocrit, body weight and spleen weight of the rats kept inan alternating light-darkness cycle from 04:00 to 18:00 for light. Toseek an effective treatment time, 5-week-old rats were assigned tocontrol or treatment groups, each group consisting of 6 subgroups, eachof 5 rats, in 6 test times at 00, 04, 08, 12, 16 and 20 hours. The ratswere randomly distributed into groups such that the baseline inter-groupdifferences in body weight, blood pressure and hematocrit of Epotreatment (Rx) versus saline and other treatment groups were notstatistically significant.

Blood pressure, hematocrit, and body weight were measured just beforeand immediately after the completion of a 4-week course of twice-weeklyEpo or physiological saline subcutaneous injections. Epo, Epo-bp, andαEpo-bp dosage was determined based on Epoetin study reports (4). TheEpo dose was 50 units per kg body weight, and an Epo-bp and (Epo-bp wereadministered in an amount equimolar with Epo. The erythropoietin(Epoetin) was from Amgen Company (Thousand Oaks, Calif.). Affinitypurified Epo-bp and αEpo-bp were prepared in our laboratories. Theantibodies were digested to Fab fragments, and the Fab antibodies werepurified.

For blood pressure measurement, the femoral artery was cannulated underpentobarbital (50 mg/kg) anesthesia. At the end of the study, spleenswere weighed and photographed. The weights of the brain, heart, aorta,and L- and R-kidneys were also obtained.

Ligand Binding Site in Progenitor Cells and Detection of Epo and EpoR:

We developed αEpo-bp in sheep innoculated with Epo-bp every 3-4 week for3 months. After collecting serum, the antibodies were purified anddigested with papain to generate Fab antibodies. The Fab were purified.Fab were fluorescein labeled according to the manufacturer'sdescription. These materials were used to detect Epo receptor in bloodand/or tissue samples. Negative control cells had no antibodies addedand positive control cells had Fab from IgG of preimmune serum. To testfor antibody binding sites (Epo receptor) bone marrow cells were washedin PBS and dispensed at 1-3×10³ cells per well in round-bottomed tubesand centrifuged into a pellet at 500 g for 2 minutes. Supernatant wasremoved and 100 μl of fluorescein-conjugated Fab antibodies were added.After mixing well, the mixture was incubated on ice for 30 min. Thecells were washed three times by adding 400 μl of buffer containing 1%FCS and 0.01% NaN₃ in PBS and centrifuged at 500 g for 2 minutes toremove supernatant. The cells were resuspended in a total volume of upto 50 μl of PBS and analyzed under an inverted fluorescence microscope.

Enzyme immunoassay (EIA) was used to detect and measure the levels ofEpo, Epo-bp, and antibodies against Epo, and Epo-bp in untreated humansubjects. EIA microplates were coated with 2 μg/well of anti-Epo todetect Epo and 2 μg/well of anti-Epo-bp antibody (αEpo-bp) to detectEpo-bp. To detect circulating anti-Epo and anti-Epo-bp antibodies, wellswere coated with 200 μl of 1:10 diluted serum or plasma in PBS, pH 7.4.Plates were incubated at room temperature for 30 minutes or at 4° C.overnight. After coating the plates with antibody or serum, wells werewashed 3 times with 200 μl/well PBST (0.05% TWEEN 20 in PBS).Nonspecific binding sites were blocked with 200 μl/well 1% BSA in PBSTfor 30 min at room. temperature. Wells were washed 3 times with 200μl/well PBST. To detect bound antigen, peroxidase-streptavidin label wasattached to Fab anti-Epo (for detecting Epo), Epo (for detectinganti-Epo antibodies), Fab anti-Epo-bp (for detecting Epo-bp), and Epo-bp(for detecting anti-Epo-bp) in our laboratory. Two micrograms of theappropriate peroxidase-streptavidin-labeled protein in 200 μl PBST wasadded per well . The wells were washed 3 times with 200 μl PBST. Asolution (160 μl) of o-phenylenediaminedihydrochloride (OPD) in citratebuffer was added to each well. (The solution contained 10 mg/ml in 24 mMcitrate, 51 mM Na₂HPO₄, pH 6.0, with 0.4 ml of 3% H₂O₂ added to 100 mlof solution immediately before use.) The reaction was stopped by adding40 μl of 5M NaOH, and the absorbance was measured at 405 nm.

Statistics:

Data were analyzed by two-tailed Student's t test, the cosinor methodand the linear least square rhythmometry (21), allowing variation as afunction of the data. Data are expressed as mean±SEM. A p value of lessthan 0.05 was considered significant.

Results

In Table 1, before treatment, the inter-group differences for bloodpressure, hematocrit, and body weight in all treatment groups were notstatistically significant. Overall, body weight was lowered by Epocompared to control (295 vs. 313 grams, p <0.01). The referencecircadian blood pressure differences in Epo treatment versus control,Epo-bp, and αEpo-bp (Fab antibody against Epo-bp) treatment groupsbefore treatment were not statistically significant (87±2.8 vs.88.8±3.4, 88.7±2.5, 84.3±2.3 mm Hg). After treatment, the circadianblood pressure was significantly increased in the Epo treated group. Thegroup comparisons between Epo treatment versus control, Epo-bp, andαEpo-bp treatment groups were as follows: 136.2±2.3 vs. 116.2±1.7,118.4±2.1 and 116.6±2.1 mm Hg, respectively, each p<0.0001. When Epo-bpor αEpo-bp was given along with Epo, however, blood pressure wasmaintained at similar levels to the saline control group: 118.3±1.7 inthe Epo-bp+Epo group and 121.0±2.0 mm Hg in the αEpo-bp+Epo treatmentgroup, which were significantly lower than that of the Epo-treated group(136.2±2.3), each p<0.0001. TABLE 1 Overall Effects upon Circadian BodyWeight, Blood Pressure, Hematocrit and Other Organ Systems in VariousTreatments. Group (Rx) {overscore (y)} before Rx (all group n = 30) BW(g) BP (mm Hg) Hct (%) Control (Saline) vs. 80.1 ± 1.7 88.8 ± 3.4 36.2 ±0.7 Epo 80.2 ± 1.4 87.1 ± 2.8 37.0 ± 0.6 Epo-bp 81.6 ± 1.5 88.7 ± 2.536.5 ± 0.7 αEpo-bp 81.2 ± 1.3 84.3 ± 2.3 36.1 ± 0.4 Epo + Epo-bp 81.0 ±1.0 84.3 ± 3.4 36.3 ± 0.6 Epo + αEpo-bp 79.4 ± 1.5 88.9 ± 2.6 37.1 ± 0.4{overscore (y)} after Rx BW (g) BP (mm Hg) Hct (%) SW (g) Brain W (g)Control (Saline) vs. 312.8 ± 4.9 116.2 ± 1.7 42.7 ± 0.8 0.86 ± 0.03 1.82± 0.01 Epo 294.9 ± 4.2* 136.2 ± 2.3*** 61.6 ± 1.3*** 1.58 ± 0.07*** 1.77± 0.02* Epo-bp 312.1 ± 3.9 118.4 ± 2.1 43.9 ± 0.6 0.89 ± 0.02 1.80 ±0.02 αEpo-bp 305.0 ± 4.9 116.6 ± 2.1 44.1 ± 0.7 0.85 ± 0.02 1.80 ± 0.01Epo + Epo-bp 303.4 ± 3.6 118.3 ± 1.7 58.0 ± 1.1*** 1.62 ± 0.05*** 1.77 ±0.02* Epo + αEpo-bp 298.4 ± 4.4 121.0 ± 2.0 59.1 ± 1.1*** 1.79 ± 0.07***1.76 ± 0.01** Epo vs. 294.9 ± 4.2 136.2 ± 2.3 61.6 ± 1.3 1.58 ± 0.071.77 ± 0.02 Epo-bp 312.1 ± 3.9* 118.4 ± 2.1*** 43.9 ± 0.6*** 0.89 ±0.02*** 1.80 ± 0.02 αEpo-bp 305.0 ± 4.9 116.6 ± 2.1*** 44.1 ± 0.7***0.85 ± 0.02*** 1.80 ± 0.01 Epo + Epo-bp 303.4 ± 3.6 118.3 ± 1.7*** 58.0± 1.1‡ 1.62 ± 0.05 1.77 ± 0.02 Epo + αEpo-bp 298.4 ± 4.4 121.0 ± 2.0***59.1 ± 1.1 1.79 ± 0.07 1.76 ± 0.01 {overscore (y)} after Rx Heart W (g)Aorta W (g) R-Kidney W (g) L-Kidney W (g) Control (Saline) vs. 1.03 ±0.02 0.046 ± 0.002 1.107 ± 0.02 1.092 ± 0.02 Epo 0.93 ± 0.02** 0.046 ±0.002 1.076 ± 0.02 1.084 ± 0.03 Epo-bp 1.03 ± 0.02 0.046 ± 0.002 1.106 ±0.02 1.083 ± 0.03 αE-Epo-bp 1.04 ± 0.02 0.044 ± 0.002 1.112 ± 0.02 1.099± 0.02 Epo + Epo-bp 0.96 ± 0.02* 0.046 ± 0.002 1.098 ± 0.02 1.073 ± 0.02Epo + αEpo-bp 0.99 ± 0.02 0.046 ± 0.002 1.084 ± 0.02 1.044 ± 0.02 Epovs. 0.93 ± 0.02 Epo-bp 1.03 ± 0.02** αE-Epo-bp 1.04 ± 0.02** Epo +Epo-bp 0.96 ± 0.02 Epo + αEpo-bp 0.99 ± 0.02*Rx: treatment;n = number of rats (30 rats in each group);{overscore (y)}: 24-h average;αEpo-bp = anti-Epo-bp antibody;*p < 0.01;**p < 0.001;***p < 0.0001;‡p < 0.05;g = gram;BW = body weight;BP = blood pressure;Hct = hematocrit;SW = spleen weight;W = weight;R = right;L = left

Epo treatment increased hematocrit markedly overall as compared to thesaline, Epo-bp or αEpo-bp groups (61.6 vs. 42.7, 43.9 and 44.1%,respectively) and at each of the 6 test times, all p<0.0001.Administering Epo-bp or αEpo-bp together with Epo had almost no effecton the Epo-induced hematocrit increase (61.6% hematocrit in Epo vs.58.0% in Epo +Epo-bp and 59.1% in Epo+αEpo-bp Rx). But, significantly,both Epo-bp and (Epo-bp almost eliminated the Epo-induced blood pressurerise (136.2 mm Hg in the Epo-treated group, vs. 116.2 in saline control,118.3 for Epo +Epo-bp, and 121.0 in Epo +αEpo-bp). Thus, both Epo-bp andαEpo-bp protected the rats from the blood pressure rise caused by Epotreatment.

Splenomegaly characterized each rat in the Epo-treated group (spleenweight overall 1.58 vs. 0.86 for saline, 0.89 for Epo-bp, and 0.85 gramsfor αEpo-bp, each p<0.000 1). Administering Epo-bp or (Epo-bp togetherwith Epo did not affect the splenomegaly. Brain and heart weights weresignificantly lower in the Epo Rx group as compared to all other groups,although the aorta and kidney weights were similar in each group.

FIG. 1 shows circadian blood pressures in all group comparisons in bargraphs +standard errors (SEM). The Epo-treated group had a significantlyincreased blood pressure as compared to all other 5 groups, eachp<0.0001. FIG. 2 shows circadian fluctuations of blood pressure in MESOR(about 24-h mean), amplitude and acrophase (peak time) in each treatmentgroup. Epo treatment increased circadian blood pressure (MESOR)significantly as compared to all other groups (all p<0.0001), althoughall group amplitude comparisons were not significantly different. Aftertreatment, the peak time in the Epo-treated rats was shifted to thedaytime as compared to control, Epo-bp or αEpo-bp treatment groups(19:40 vs. 04:08, 05:44, 05:16, respectively). It is an obvious shiftchange from the night to the daytime peak with Epo treatment in thisnocturnal animal. When Epo-bp or αEpo-bp was given together with Epo,the shift change still remained in the same daytime range as in theEpo-alone treatment group (14:48, 19:20, respectively), although theEpo-bp+Epo and αEpo-bp+Epo groups' blood pressure levels were similar tothe control group.

Table 2 summarizes the circadian variations of body weight, bloodpressure, hematocrit and spleen weight in the 6 subgroups after Epo,Epo-bp and αEpo-bp treatments. The body weight difference betweenEpo-treated rats and any other treatment group was not statisticallysignificant among the 6 test times. A significantly increased bloodpressure in the Epo treated group was detected at 12, 16, 20 and 00hours, but not at 04 or 08 hours as compared to control, Epo-bp and αEpobp Rx groups. Epo treatment increased hematocrit markedly overall and ateach of the 6 test times as compared to control, Epo-bp and αEpo-bp Rxgroups, all p<0.0001. The spleen weights were significantly higher inthe Epo-treated group rats than those of the control, Epo-bp and αEpo-bpgroups at all time points, although the body weight was lower at eachtime comparison. TABLE 2 Circadian Variations of Body Weight, BloodPressure, Hematocrit and Spleen Weight in Various Treatments Group (Rx)0400 0800 1200 1600 2000 0000 BW (gram): Saline vs.  313 ± 112  305 ± 09 324 ± 18  308 ± 13  310 ± 10  317 ± 13 Epo  305 ± 13  294 ± 07  294 ±05  290 ± 05  295 ± 14  291 ± 14 Epo-bp  314 ± 11  310 ± 06  303 ± 04 312 ± 10  319 ± 11  319 ± 13 αEpo-bp  314 ± 10  297 ± 20  308 ± 13  299± 06  293 ± 05  320 ± 12 Epo + Epo-bp  297 ± 10  300 ± 04  301 ± 09  308± 11  301 ± 11  313 ± 09 Epo + αEpo-bp  296 ± 13  286 ± 06  279 ± 04* 304 ± 12  305 ± 05  320 ± 13 BP (mm Hg): Saline vs.  116 ± 5.8  120 ±4.6  117 ± 3.7  108 ± 1.0  119 ± 3.2  118 ± 4.1 Epo  131 ± 7.6  131 ±4.8  139 ± 3.9*  128 ± 8.1‡  140 ± 6.3*  137 ± 6.2‡ Epo-bp  118 ± 4.5 122 ± 5.5  118 ± 3.6  115 ± 4.0  118 ± 6.1  120 ± 7.7 αEpo-bp  113 ±5.7  122 ± 5.1  117 ± 4.7  112 ± 3.4  113 ± 4.6  122 ± 6.8 Epo + Epo-bp 114 ± 2.2  121 ± 3.1  118 ± 6.9  121 ± 4.5‡  119 ± 4.0  117 ± 3.8 Epo +αEpo-bp  116 ± 6.7  121 ± 4.1  120 ± 6.2  120 ± 5.0‡  127 ± 5.6  122 ±1.4 Epo vs. Epo-bp  118 ± 4.5  122 ± 5.5  118 ± 3.6*  115 ± 4.0‡  118 ±6.1‡  120 ± 7.7 αEpo-bp  113 + 5.7  122 ± 5.1  117 ± 4.7*  112 ± 3.4‡ 113 ± 4.6*  122 ± 6.8 Epo + Epo-bp  114 ± 2.2  121 ± 3.1  118 ± 6.9‡ 121 ± 4.5  119 ± 4.0‡  117 ± 3.8‡ Epo + αEpo-bp  116 ± 6.7  121 ± 4.1 120 ± 6.2‡  120 ± 5.0  127 ± 5.6  122 ± 1.4‡ Hct (%): Saline vs.   42 ±2.6   41 ± 2.3   42 ± 1.6   44 ± 0.5   45 ± 1.4   43 ± 3.0 Epo   60 ±4.5*   64 ± 2.2***   66 ± 2.7***   65 ± 1.5***   61 ± 3.8*   64 ± 1.5**Epo-bp   45 ± 1.4   45 ± 1.6   44 ± 1.1   41 ± 2.2   45 ± 0.6   43 ± 1.9αEpo-bp   47 ± 1.0   45 ± 0.8   43 ± 0.8   43 ± 3.2   43 ± 2.2   44 ±0.9 Epo + Epo-bp   58 ± 1.9**   62 ± 1.8***   60 ± 2.9**   62 ± 2.5***  54 ± 3.2‡   53 ± 3.5 Epo + αEpo-bp   61 ± 2.0***   64 ± 1.9***   60 ±2.7***   57 ± 4.0‡   57 ± 3.2*   55 ± 1.8* SW (gram): Saline vs. 0.88 ±0.1 0.82 ± 0.1 0.88 ± 0.1 0.96 ± 0.1 0.73 ± 0.1 0.92 ± 0.1 Epo 1.65 ±0.2* 1.70 ± 0.2** 1.69 ± 0.1*** 1.63 ± 0.1* 1.37 ± 0.1* 1.23 ± 0.1‡Epo-bp 0.87 ± 0.0 0.87 ± 0.0 0.87 ± 0.0 0.94 ± 0.1 0.97 ± 0.1‡ 0.83 ±0.1 αEpo-bp 0.82 ± 0.0 0.92 ± 0.1 0.87 ± 0.0 0.80 ± 0.1 0.79 ± 0.1 0.89± 0.0 Epo + Epo-bp 1.50 ± 0.1** 1.58 ± 0.1*** 1.67 ± 0.1*** 1.62 ± 0.2*1.48 ± 0.1*** 1.86 ± 0.1*** Epo + αEpo-bp 1.69 ± 0.1*** 1.54 ± 0.2**1.53 ± 0.1*** 1.80 ± 0.1** 1.90 ± 0.2*** 2.27 ± 0.3** Epo vs. Epo-bp0.87 ± 0.0** 0.87 ± 0.0** 0.87 ± 0.0*** 0.94 ± 0.1* 0.97 ± 0.1 0.83 ±0.1‡ αEpo-bp 0.82 ± 0.0** 0.92 ± 0.1* 0.87 ± 0.0*** 0.80 ± 0.1** 0.79 ±0.1* 0.89 ± 0.0* Epo + Epo-bp 1.50 ± 0.1 1.58 ± 0.1 1.67 ± 0.1 1.62 ±0.2 1.48 ± 0.1 1.86 ± 0.1* Epo + αEpo-bp 1.69 ± 0.1 1.54 ± 0.2 1.53 ±0.1 1.80 ± 0.1 1.90 ± 0.2 2.27 ± 0.3*Rx = Treatment;n = 5 rats in each subgroup;*p < 0.01;**p < 0.001;***p < 0.0001;‡p < 0.05;BP = Blood pressure;BW = Body weight;Hct = Hematocrit;SW = Spleen weight.

FIG. 3 shows circadian hematocrit comparisons. There was not only anincreased hematocrit but also the peak time of hematocrit shifted fromnight (20:15) to a late morning hour (1 1:16) with Epo-treatment. In thegraph, groups of a (control), c (Epo-bp) and d (αEpo-bp) are located inthe dark cycle plane, while groups of b (Epo), e (Epo +Epo-bp), and f(Epo±αEpo-bp) are located in the light cycle plane. Again, it is anobvious shift change from the night to the daytime peak in Epo Rx inthis nocturnal animal. MESOR comparisons in % hematocrit in Epo (61.6)versus control (42.7), Epo-bp (43.9) and αEpo-bp (44.1) treatment wereall statistically significant in each time point comparison (eachp<0.0001). The amplitudes of the circadian peak-to-peak differences inhematocrit were not significantly different between the treatmentgroups. But the amplitudes were larger in the Epo-treated groups than inthe groups that did not receive Epo (2.40 in Epo, 4.41 in Epo+Epo-bp,and 3.59 in Epo±αEpo-bp, versus 1.65 in control, 1. 13 in Epo-bp and1.73 in αEpo-bp).

In FIG. 4, splenomegaly (a, b and c) characterized each Epo treated ratwhen compared with the saline treated rats (FIG. 4, panel d). The spleenweight was significantly higher in Epo treated rats, as compared tothose of control, Epo-bp and αEpo-bp Rx groups (Tables 1 and 2).

The results suggest that the time of the Epo treatment, with or withoutEpo-bp and/or αEpo-bp treatment may be important. Epo-bp and αEpo-bpprotect against the Epo-caused blood pressure rise, while not reducingthe Epo-increased hematocrit levels.

Epo dose in clinical use should be reevaluated to prevent furthersystemic and local adverse effects, such as high blood pressure andother organ damages.

The binding sites of blood cell progenitors were identified using Epo-bpand antibodies against it. Fluorescein-labeled αEpo-bp was used tovisualize receptor sites of bone marrow progenitor cells. No receptorswere detected with fluorescein-labeled preimmune Fab, or in negativecontrol cells. But labeled αEpo-bp did detect binding sites onmegakaryocytes, erythroblasts, normoblasts, and myeloblasts (data notshown).

The levels of Epo, Epo-bp, and antibodies against Epo and Epo-bp weremeasured in serum and plasma in untreated humans by enzyme immunoassay(EIA The EIA results are presented in FIG. 5. Optical density (OD) ofeach measurement is presented as the mean ±SEM of 8-14 individualsamples in duplicates. The OD values presented in FIG. 5 were calculatedby subtracting the OD value of the blanks from the OD of each sample.Serum and plasma Epo and Epo-bp OD values were similar to each other:0.308±0.026 serum Epo, 0289±0.022 serum Epo-bp, 0.289±0.028 plasma Epo,and 0.299±0.015 plasma Epo-bp. The plasma level of anti-Epo-bp antibodywas significantly lower than those of the other three antibodycategories: 0.058±0.008 serum αEpo, 0.052±0.006 serum αEpo-bp,0.054±0.013 plasma αEpo, and 0.031±0.004 plasma αEpo-bp. Serum αEpo andαEpo-bp levels were similar but the concentration of plasma αEpo-bp wassignificantly lower than the concentration of serum αEpo, serum αEpo-bp,or plasma αEpo (p<0.025). The Epo and Epo-bp values were converted withknown Epo concentrations prepared as controls in the same plate tomU/ml. The converted values in mU/ml were 25.4±2.17 mU serum Epo,24.2±2.35 mU plasma Epo; 24.2±1.84 mU serum Epo-bp, 25.0±1.26 mU plasmaEpo-bp. This assay method is simple and more sensitive than theradioimmunoassay (17.7±6.3 mU/ml of Epo) and gives a much smaller SEM.Furthermore, the materials used in the preparation are moreenvironmentally friendly than radioactive or other toxic chemicals usedin conventional methods.

Discussion

As expected, the hematocrit was markedly increased overall and at eachof the 6 test times in the Epo-treated rats. In addition, we observed anincrease in blood pressure in the Epo-treated group, and splenomegalycharacterized each rat with the Epo treatment. Epo treatment not onlysignificantly increased blood pressure but also shifted the peak time ofblood pressure from the night to the daytime.

Remarkably, Epo-bp and αEpo-bp protected the rats almost completely fromthe Epo-induced rise in blood pressure, while not reducing hematocritpercent. The mechanism of this protective effect is not known. We couldspeculate, however, that Epoetin (recombinant Epo currently in clinicaluse) may induce some toxic materials in the living animal body whenrepetitively injected. Epo-bp and/or αEpo-bp might bind and eliminatethe toxic materials, since Epo-bp binds Epo or its degradation products,and αEpo-bp might also bind certain products induced by Epo treatment.

FIGS. 2 and 3 show that Epo, as well as combination treatment with Epo+Epo-bp or Epo+αEpo-bp caused a shift in the circadian time of peakblood pressure. This suggests that treatment time for treatment withEpo, Epo+Epo-bp, or Epo+αEpo-bp may markedly affect the outcome. Anindividual's genetic susceptibility to endocrine treatment, as shown bysalt susceptibility to hypertension in Dahl rats, also must beconsidered (22, 23).

The cloning of the human Epo-receptor recombinant vector JYL26 andpurification of the pure human Epo-bp and its antibodies were importantbenchmarks to allow us to visualize the ligand binding sites and toidentify the cell type where the Epo receptor is located (Lee, U.S. Pat.No. 5,843,726). To identify the ligand-binding site, we developedseveral sensitive and simple methods. These may allow us to understandthe structure of the Epo receptor, and examine the factors involved inligand binding, as well as to identify other factors involved inregulating differentiation and proliferation of the progenitor cells. Inthis study, we report the direct binding of Epo to our purified humanEpo-bp. Our Epo-bp and its antibodies are to our knowledge the firstpurified pure human Epo receptor gene products, which are characterizedin specific binding of Epo and its antibodies in nM concentrations.

The binding sites of blood progenitor cells were elaborated using Epo-bpand its antibodies. These data support the current proposal that allhuman progenitor blood cells contain Epo receptors and bind Epo. We donot know what the biophysiological mechanisms of Epo or the secondmessenger system involved in response to the Epo-Epo receptorinteraction are. The methods presented in this report will help identifydefects related to Epo or Epo receptor, and elucidate the role of Eporeceptor (EpoR) in progenitor processes and ligand binding. The resultsmay help in understanding the structural and functional relationship ofEpo-EpoR interactions in blood cell progenitors. The sensitive detectionmay help us to understand the role of the Epo-EpoR interaction in bloodcell production and diseases of blood cell production and help todevelop treatment methods for hematological malignancies and somesystemic cardiovascular diseases, such as high blood pressure.

CONCLUSIONS

Epo treatment increased hematocrit markedly overall as compared to thesaline control, Epo-bp, and anti-Epo-bp antibody (αEpo-bp) treatedgroups, and did so at each of the 6 test times, all p<0.0001. Increasedblood pressure was detected at 12, 16, 20 and 00 hours, but not at 04 or08 hours in rats treated with Epo. When Epo-bp or αEpo-bp was given inconjunction with Epo treatment, blood pressure was maintained at similarlevels to the control group. However, hematocrit levels were notsignificantly changed in Epo treatment versus Epo+Epo-bp or Epo+αEpo-bptreatment groups (61.6 vs. 58.0 or 59.1%, respectively). Thus, Epo-bpand αEpo-bp reduce or prevent the Epo-induced rise in blood pressure.

Body weight was lowered by Epo treatment. Splenomegaly characterizedeach rat in Epo treatment. Brain and heart weights were significantlylower in the Epo-treated group as compared to all other groups. Thesedata suggest that Epo dose should be reevaluated to prevent furtherorgan damage. The circadian results indicate that the time of the Epotreatment, alone or in combination of Epo-bp and/or (Epo-bp, may also beimportant.

Serum and plasma levels of Epo, Epo-bp, and antibodies against theproteins in untreated human volunteers were determined. Serum and plasmaEpo and Epo-bp levels were similar: Epo 25.4±2.17; 24.2±2.35; and Epo-bp24.2±1.84; 25.0±1.26 mU/ml, respectively. Serum αEpo and αEpo-bp levelswere similar, but the plasma αEpo-bp level was significantly lower thanthat of serum or plasma αEpo or serum αEpo-bp.

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All cited patents, patent documents, and references are herebyincorporated by reference.

1. A method of treating anemia in a mammal comprising: administeringerythropoietin (Epo) to the mammal; and administering to the mammal anagent selected from a soluble Epo-binding protein (Epo-bp), arecognition protein that binds Epo receptor on an extracellular solubleportion of the Epo receptor, and a combination thereof.
 2. The method ofclaim 1 wherein the agent is a soluble Epo-bp.
 3. The method of claim 2wherein the soluble Epo-bp comprises a fragment of a soluble portion ofa mammalian Epo receptor.
 4. The method of claim 3 wherein the solubleEpo-bp comprises a fragment of at least 30 residues of SEQ ID NO:2. 5.The method of claim 4 wherein the soluble Epo-bp is a fragment of atleast 30 residues of SEQ ID NO:2.
 6. The method of claim 2 wherein thesoluble Epo-bp comprises SEQ ID NO:2.
 7. The method of claim 6 whereinthe soluble Epo-bp is SEQ ID NO:2.
 8. The method of claim 1 wherein theagent is a recognition protein that binds Epo receptor on anextracellular soluble portion of the Epo receptor.
 9. The method ofclaim 8 wherein the recognition protein binds SEQ ID NO:2.
 10. Themethod of claim 8 wherein the recognition protein is an antibody. 11.The method of claim 10 wherein the antibody is an antibody fragment. 12.The method of claim 11 wherein the antibody fragment is Fab.
 13. Themethod of claim 1 wherein Epo and the agent are administered together.14. The method of claim 1 wherein Epo and the agent are administeredseparately.
 15. The method of claim 1 wherein the amount of agentadministered is at least equimolar with the amount of Epo administered.16. The method of claim 1 wherein the amount of agent administered isabout equimolar with the amount of Epo administered.
 17. The method ofclaim 1 wherein the agent reduces an erythropoietin-induced bloodpressure rise in the mammal.
 18. A method of reducing hypertension in amammal receiving erythropoietin (Epo), the method comprising:administering to the mammal an effective amount of an agent selectedfrom a soluble erythropoietin-binding protein (Epo-bp), a recognitionprotein that binds Epo receptor on an extracellular soluble portion ofthe Epo receptor, and a combination thereof.
 19. A pharmaceuticalcomposition comprising: erythropoietin (Epo); and an agent selected froma soluble Epo-binding protein (Epo-bp), a recognition protein that bindsEpo receptor on an extracellular soluble portion of the Epo receptor,and a combination thereof.
 20. The pharmaceutical composition of claim19 wherein the recognition protein is an antibody against SEQ ID NO:2.21. The pharmaceutical composition of claim 19 wherein the Epo-bp is SEQID NO:2.
 22. A pharmaceutical composition comprising: a recognitionprotein that binds erythropoietin (Epo) receptor on an extracellularsoluble portion of the Epo receptor.
 23. The pharmaceutical compositionof claim 22 wherein the recognition protein is an antibody against SEQID NO:2.
 24. A pharmaceutical composition comprising: a solubleerythropoietin-binding protein (Epo-bp).
 25. The pharmaceuticalcomposition of claim 24 wherein the Epo-bp is SEQ ID NO:2.
 26. Thepharmaceutical composition of claim 24 wherein the Epo-bp is a productof a process comprising: expressing a fusion protein consisting of: (a)a first polypeptide segment having an amino terminus and a carboxylterminus, said segment having SEQ ID NO:7 at its carboxyl terminus; and(b) a second polypeptide segment consisting of SEQ ID NO:2, the secondpolypeptide segment covalently coupled to the carboxyl terminus of thefirst polypeptide segment; and cleaving the fusion protein withthrombin.